The present invention relates to a novel compound and derivatives thereof, to processes for their production, to pharmaceutical formulations containing them, to their use in medical therapy, particularly in the treatment of bacterial and fungal infections, and also to their use as immunosuppressants.
Rapamycin is a known compound and was first isolated as an extract of the fungus Streptomvces hygroscopicus and reported to have antifungal activity (British Patent 1436447). Subsequently rapamycin has been implicated as an immunosuppressant (Martel R. R. et al Can. J. Physiol. Pharmacol. 55, 48-51, 1977).
A large number of microorganisms have been found to produce a variety of metabolites which have subsequently been isolated and have been shown to possess useful therapeutic properties. One such compound is 29-desmethylrapamycin. This is believed to be a novel compound and has been found to have useful antifungal activity and also immunosuppressant properties.
Accordingly the present invention provides 29-desmethylrapamycin and derivatives thereof.
The invention in a second aspect, further provides a process for the production of 29-desmethylrapamycin which comprises cultivating a producing microorganism and subsequently isolating 29-desmethylrapamycin or derivatives thereof.
29-desmethylrapamycin is believed to have the following structure: 
It has the following characteristics:
i) it has an apparent molecular weight of 899 by fast atom bombardment (FAB) mass spectroscopy,
ii) it may be obtained by the cultivation of a microorganism from the genus Streptomyces,
iii) 13CNMR spectroscopy reveals 50 carbons in the molecule,
iv) it shows antifungal activity against Candida albicans. 
v) it shows immunosuppressant properties.
29-desmethylrapamycin may be obtained by the cultivation of a producing organism and the recovery of it or a derivative thereof from the culture.
The term xe2x80x98cultivationxe2x80x99 (and derivatives of that term) as used herein means the deliberate aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen, sulphur and mineral salts. Such aerobic growth may take place in a solid or semi-solid nutritive medium, or in a liquid medium in which the nutrients are dissolved or suspended. The cultivation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically defined.
It has been found that suitable microorganisms for use in the cultivation process according to the invention include bacterial strains belonging to the genus Streptomyces which are capable of elaborating 29-desmethylrapamycin. It has further been found been isolated from nature and also mutants thereof.
The term xe2x80x98mutantxe2x80x99 as used herein includes any mutant strain which arises spontaneously or through the effect of an external agent whether that agent is applied deliberately or otherwise. Suitable methods of producing mutant strains including those outlined by H. I. Adler in xe2x80x98Techniques for the Development of Microorganismsxe2x80x99 in xe2x80x98Radiation and Radioisotopes for Industrial Microorganismsxe2x80x99, Proceedings of a Symposium, Vienna, 1973, page 241, International Atomic Energy Authority, and these include:
(i) Ionizing radiation (e.g. X-rays and xcex3-rays), u.v. light, u.v. light plus a photosensitizing agents (e.g. 8-methoxypsoralen), nitrous acid, hydroxylamine, pyrimidine base analogues (e.g. 5-bromouracil), acridines, alkylating agents (e.g. mustard gas, ethyl-methane sulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and
(ii) Genetic techniques, including, for example, recombination, transformation, transduction, lysogenisation, lysogenic conversion, protoplast fusion and selective techniques for spontaneous mutants.
Using the methods of Becker B. Lechevalier M. P., Gordon R. E., Lechevalier H. A., 1964, Appl. Microbiol. 12, 421-423 and Williams S. T., Goodfellow M, Wellington E. M. H., Vickers J. C., Alderson. G., Sneath P. H. A., Sackin M. J., and Mortimer M. 1983 J. Gen. Microbiol. 129, 1815-1830, Sp. NCIB 40319 has been identified as a previously unreported, atypical, strain of Streptomyces and therefore also forms a part of the present invention, particularly in biologically pure form. It has been deposited at the National Collections of Industrial and Marine Bacteria Ltd. (N.C.I.B), Aberdeen, Scotland under number 40319 on Sep. 14, 1990.
Strain NCIB 40319 has been characterised as follows:
The method of whole-cell amino acid analysis was that described by Becker et al (1964). Identification media used for the characterisation of the culture were as described by Williams et al (1983). In addition, starch casein agar (Waksman S. A., 1961. The Actinomycetes Vol. 2 Williams and Wilkins Co. Baltimore ppl-363) was used for the morphological description of the culture.
The microorganism was characterised by inoculating agar blocks from a well grown plate into Y broth (see Table 1) and incubating for three days at 28xc2x0 C. on a shaker. It was then centrifuged for 20 minutes at 3660 rpm, washed twice with distilled water, then finally resuspended in phosphate buffered saline (Dulbecco A). This inoculum was plated onto media commonly used for the identification of members of the Actinomycetales as above. Plates were incubated at 28xc2x0 C. and the results were read at varying times but most were commonly taken at 14 days. The colours are described in common terminology but exact colours were determined by comparison with colour chips from the Methuen Handbook of colour (3rd Edn).
Results:
Cell Wall analysis
The whole-cell hydrolysates contained LL-diaminopimelic acid. The observations of growth and appearance of the organism were as follows:
Yeast extract-Malt extract Aear (ISP 2 Difco)
Growth good, cream 22a), with a white powdery centre. Colonies raised and rather wrinkled, no sporulation.
Inorganic Salts Starch Agar (ISP4 Difco)
Growth good, white with pale grey to grey (11b, 11c) aerial mycelium. Colonies quite flat with slightly raised centre. Reverse cream (22a).
Glycerol Asparagine Agar (S. A. Waksman, 1961, p328). medium No. 2.
Growth moderate to good, white with grey centre (11d).Colonies flat, reverse cream (22a).
Starch Mineral Salts Agar
Growth very poor, opaque small colonies. No aerial mycelium.
Starch Casein Agar
Growth good, white with light grey to grey central area (11c, 11d), occasional small patch of white non-sporulating mycelium in grey sporulating areas. Tiny colourless droplets over the grey areas. Colonies fairly flat and gently rounded. Small black hygroscopic patches may occur after 4 weeks incubation.
Morphological Properties
These were observed after two weeks incubation on starch casein agar: spore mass in grey colour-series; spore chains in section spirales, tightly coiled or slightly open, of small diameter, generally 2-6 coils, occasionally more, may aggregate into hygroscopic masses. There was no fragmentation of vegetative mycelium.
Biochemical Properties
See Table 2 for full details. In summary, melanin not produced; nitrate not reduced to nitrite in organic nitrate broth; H2S produced in peptone-yeast extract iron broth; no growth on inhibitors; degradation only of arbutin, antibiosis only against Bacillus subtilis. Carbohydrate utilization glucose, cellobiose, fructose, inositol, mannitol, raffinose, rhamnose and xylose. Nitrogen sources used: asparagine, histidine and hydroxyproline, a-amino-butyric acid used only slightly.
Determination of Identification Scores
These were obtained using the Matiden program (Sneath P. H. A., 1979. Computers and Geosciences 5 195-213) which provides the best identification scores for known or unknown strains against the percent probability matrix of Williams et al (1983). Willcox Probabilityxe2x80x94the nearer the score reaches 1.0, the better is the fit of an unknown with a group in the matrix (scores of  greater than 0.85 acceptable) Taxonomic distancexe2x80x94low scores indicate relatedness (scores  less than 0.3 acceptable). The organism had acceptable identification scores with cluster 32 (violaceoniger) which contains Streptomyces hygroscopicus species.
Conclusion
The culture is characterised by the grey spores in mass, the negative melanin reaction and the spores which are arranged in spirally coiled chains. The spore chains may coalesce into hygroscopic masses. The culture utilised a wide range of carbohydrate sources. The whole-cell hydrolysate indicates the presence of LL-diaminopimelic acid.
The fermentation medium for cultivating sp. NCIB 40319 suitably contains sources of assimilable carbon and assimilable nitrogen together with inorganic salts. Suitable sources of nitrogen include yeast extract, soyabean flour, meat extract, cottonseed, flour, malt, distillers dried solubles, amino acids, protein hydrolysates and ammonium and nitrate nitrogen. Suitable carbon sources include glucose, lactose, maltose, starch and glycerol. Suitably the culture medium also includes alkali metal ions (for example, sodium), halogen ions (for example, chloride), and alkaline earth metal ions (for example calcium and magnesium), as well as trace elements such as iron and cobalt.
The cultivation may suitably be effected at a temperature of about 20 to 35xc2x0 C., advantageously 20 to 30xc2x0 C., and the culture may suitably be harvested up to 7 days, advantageously about 3 to 5 days, after the initiation of fermentation in order to give an optimum yield of the desired product.
The desired product or a derivative thereof may then be isolated from the culture medium and worked up and purified using conventional techniques for such compounds. All such isolation and purification procedures may conveniently be effected at cool to ambient temperature, for example at a temperature within the range of from 4 to 40xc2x0 C., conveniently from 20 to 35xc2x0 C.
The desired compound may readily be identified in a routine manner by testing for antifungal activity and/or by monitoring the h.p.l.c. retention time.
Suitably, the separation procedure may include a high-performance liquid chromatography step, preferably as the last step. Elution may be effected using aqueous methanol.
29-desmethylrapamycin and its derivatives may be crystalline or non-crystalline and, if crystalline, may optionally be hydrated or solvated.
The derivatives are preferably pharmaceutically acceptable derivatives. Derivatives may include salts with pharmaceutically acceptable counter ions.
The compounds according to the invention are suitably provided in substantially pure form, for example at least 50% pure, suitable at least 60% pure, advantageously at least 75% pure, preferably at least 85% pure, more preferably at least 95% pure, especially at least 98% pure, all percentages being calculated as weight/weight. An impure or less pure form of a compound according to the invention may, for example, be used in the preparation of a more pure form of the same compound or of a related compound (for example a corresponding derivative) suitable for pharmaceutical use.
29-desmethylrapamycin and its pharmaceutically acceptable derivatives have antifingal and immunosuppressant properties and are useful for the treatment of fungal infections in animals, especially mammals, including humans, in particular humans and domesticated animals (including farm animals). The compounds may be used for the treatment of topical fungal infections in man caused by, among other organisms, species of Candida, Trichophyton, Microsporum or Epidermophyton or in mucosal infections caused by Candida albicans (e.g. thrush and vaginal candidiasis). They may also be used in the treatment of systemic fungal infections caused by, for example Candida albicans, Cryptococcus neoforma, Asperaillus fumigatus, Coccidiodes, Paracocciciodes, Histoplasma or Blastastomyces spp. They may also be of use in treating eumycotic mycetoma, chromoblastomycosis and phycomycosis.
The compound of the invention is active as an immunomodulatory agent. The term xe2x80x9cimmunomodulatory agentxe2x80x9d means that the compound of the invention is capable of inducing immune suppression by inhibiting T (and B) cell responses in vitro and/or by producing a statistically significant decrease in the inflammation system response medicated secondary lesion in the adjuvant induced arthritis. Indications for therapy using an immunomodulatory agent include, but are not limited to, the treatment of the following disease states:
rheumatoid arthritis
systemic lupus erythematosis
multiple sclerosis
acute transplantation/graft rejection
myasthenia gravis
progressive systemic sclerosis
multiple myeloma
atopic dermatitis
hyperimmunoglobulin E
hepatitis B antigen negative chronic active hepatitis
Hashimoto""s thyroiditis
Familial Mediterranean fever
Grave""s disease
autoimmune hemolytic anemia
primary biliary cirrhosis
inflammatory bowel disease
insulin dependent diabetes mellitus
Accordingly the invention provides 29-desmethylrapamycin or derivative for use in medical therapy. Preferably for use as an antifungal agent or an immunomodulatory agent.
The invention further provides a method of treating a human or animal suffering from a fungal infection by the administration of an effective amount of 29-desmethylrapamycin or derivative thereof.
Moreover, the invention provides a method of treating a human or animal in need of immunomodulation by administration of an effective amount of 29-desmethylrapamycin or derivative thereof.
The invention further provides a pharmaceutical composition comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable diluent or carrier. The composition is preferably for human use in tablet, capsule, injectable or cream form.
For human use 29-desmethylrapamycin or derivatives thereof can be administered alone, but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they may be administered orally in the form of a tablet containing such excipients as starch or lactose, or in a capsule or ovule either alone or in admixture with excipients, or in the form of an elixir or suspension containing a flavouring or colouring agent. They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile solutions which may contain other substances, for example, enough salts or glucose to make the solution isotonic.
For oral and parenteral administration to human patients suffering from a fungal infection, it is expected that the daily dosage level of the antifungal compounds of formula (I) will be from 0.1 to 10 mg/kg (in divided doses) when administered by either the oral or parenteral route. Thus tablets or capsules of the compounds can be expected to contain from 5 mg to 0.5 g of active compound for administration singly or two or more at a time as appropriate. The physician in any event will determine the actual dosage which will be most suitable for an individual patient and will vary with the age,,weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Equally for a human patient in need of immunomodulation the daily parenteral or oral dosage regimen for the compound or derivative thereof will preferably be from 0.1 mg/kg to 30 mg/kg.
No unacceptable toxicological effects are expected when the compound is administered in the above mentioned dosage ranges.
The compounds and compositions according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antifungal or immunomodulatory agent.
The compounds and tablets and capsules for oral administration may be in unit dosage form, and may contain conventional excipients including, for example, binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; and pharmaceutically acceptable wetting agents, for example sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice.
Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain conventional additives, including, for example, suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters (for example glycerine), propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavouring and colour agents.
Compositions according to the invention intended for topical administration may, for example, be in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, impregnated dressings, and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
Compositions according to the invention may be formulated as suppositories, which may contain conventional suppository bases, for example cocoa-butter or other glycerides.
Compositions according to the invention intended for parenteral administration may conveniently be in fluid unit dosage forms, which may be prepared utilizing the compound and a sterile vehicle, propyleneglycol. The compound, depending on the vehicle and concentration used, may be either suspended or dissolved in the vehicle. Parenteral suspensions may be prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilisation cannot be accomplished by filtration. The compound may instead be sterilised by exposure to ethylene oxide before being suspended in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in such suspensions in order to facilitate uniform distribution of the compound.